Characterization of cells susceptible to SARS-COV-2 and methods for detection of neutralizing antibody by focus forming assay
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Abstract
The SARS-CoV-2 outbreak and subsequent COVID-19 pandemic have highlighted the urgent need to determine what cells are susceptible to infection and for assays to detect and quantify SARS-CoV-2. Furthermore, the ongoing efforts for vaccine development have necessitated the development of rapid, high-throughput methods of quantifying infectious SARS-CoV-2, as well as the ability to screen human polyclonal sera samples for neutralizing antibodies against SARS-CoV-2. To this end, our lab has adapted focus forming assays for SARS-CoV-2 using Vero CCL-81 cells, referred to in this text as Vero WHO. Using the focus forming assay as the basis for screening cell susceptibility and to develop a focus reduction neutralization test. We have shown that this assay is a sensitive tool for determining SARS-CoV-2 neutralizing antibody titer in human, non-human primate, and mouse polyclonal sera following SARS-CoV-2 exposure. Additionally, we describe the viral growth kinetics of SARS-CoV-2 in a variety of different immortalized cell lines and demonstrate via human ACE2 and viral spike protein expression that these cell lines can support viral entry and replication.
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SciScore for 10.1101/2020.08.20.259838: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement IACUC: Ethics statement: All animal studies were conducted in accordance with the Guide for Care and Use of Laboratory Animals of the National Institutes of Health and approved by the Saint Louis University Animal Care and Use Committee (IACUC; protocol 2771) Randomization not detected. Blinding not detected. Power Analysis not detected. Sex as a biological variable not detected. Cell Line Authentication not detected. Table 2: Resources
Antibodies Sentences Resources The primary antibody consisted of polyclonal anti-SARS-CoV-2 guinea pig sera (BEI: NR-0361) and was diluted 1:15,000 with FFA Staining Buffer (1 × PBS, 1mg/ml saponin (Sigma: 47036)). anti-SARS-CoV-2suggested: NoneT… SciScore for 10.1101/2020.08.20.259838: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement IACUC: Ethics statement: All animal studies were conducted in accordance with the Guide for Care and Use of Laboratory Animals of the National Institutes of Health and approved by the Saint Louis University Animal Care and Use Committee (IACUC; protocol 2771) Randomization not detected. Blinding not detected. Power Analysis not detected. Sex as a biological variable not detected. Cell Line Authentication not detected. Table 2: Resources
Antibodies Sentences Resources The primary antibody consisted of polyclonal anti-SARS-CoV-2 guinea pig sera (BEI: NR-0361) and was diluted 1:15,000 with FFA Staining Buffer (1 × PBS, 1mg/ml saponin (Sigma: 47036)). anti-SARS-CoV-2suggested: NoneThe secondary antibody consisted of goat anti-mouse conjugated horseradish peroxidase (Sigma: A-7289) diluted 1:5,000 in FFA Staining Buffer. anti-mousesuggested: NoneExperimental Models: Cell Lines Sentences Resources Virus was passaged two times in Vero E6 cells (ATCC® CRL-1586™) before clarification by centrifugation (3000 rpm for 30 min) and storage at −80°C until further use. Vero E6suggested: NoneSHHC17 cells were gifted by K. Toth. SHHC17suggested: NoneFocus forming assay: One day prior to the assay, Vero WHO cells were plated in a 96 well flat bottom tissue culture treated plate. Vero WHOsuggested: ECACC Cat# 88020401, RRID:CVCL_JF53)Media was then removed from the 96-well flat bottom plate containing the Vero WHO cell monolayer and replaced with 100μL per well of diluted samples. Verosuggested: NoneSoftware and Algorithms Sentences Resources The total peak area under the curve (AUC) was calculated using GraphPad Prism 8. GraphPad Prismsuggested: (GraphPad Prism, RRID:SCR_002798)Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).
Results from LimitationRecognizer: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.Results from TrialIdentifier: No clinical trial numbers were referenced.
Results from Barzooka: We did not find any issues relating to the usage of bar graphs.
Results from JetFighter: We did not find any issues relating to colormaps.
Results from rtransparent:- Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
- Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
- No protocol registration statement was detected.
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